C.P.1.10 Molecular mechanisms underlying X-linked myotubular myopathy

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parent on blood DNA testing. By the time the result was obtained the mother was again pregnant. Due to anxiety she requested prenatal testing. This confirmed that the second baby carried the same gene alteration. The pregnancy continued and the second baby was affected in a similar fashion to the first and died at approximately the same age. The poster will outline the features of the condition in the two boys. The genetic implications from this family will be discussed and professionals need to be aware of the potential risk of germline mosasism in nemaline myopathy and include this information in family genetic counselling.

deformity in one of them. Muscle biopsies showed a disproportion of fibre size with type 1 fibres being smaller than type 2 fibres. The mutation identified was a homozygous deletion of the first nucleotide, an adenine, of the last exon of TPM3. The parents were found to be healthy mutation carriers. This mutation of the last nucleotide before the STOP codon disrupts the reading frame and causes read-through across the STOP codon. RTPCR predicts this to result in a TPM3 protein elongated by 75 amino acids. A different mutation altering the same amino acid has been published previously, but this is the first deletion to be identified in TPM3.

doi:10.1016/j.nmd.2007.06.251

doi:10.1016/j.nmd.2007.06.253

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C.P.1.09

Exercise intolerance as a presentation of adult onset nemaline (rod) myopathy Dejthevaporn, C. 1,*; Phudhichareonrat, S. 2 1 Ramathibodi Hospital Mahidol University, Medicine, Bangkok, Thailand; 2 Prasat Neurological Institute, Pathology, Bangkok, Thailand

Clinicopathological features in seven Korean patients with nemaline myopathy Choi, Y. 1,*; Na, S. 2 1 Yonsei University College of Medicine, Department of Neurology, Seoul, Republic of Korea; 2 Konyang University Hospital, Department of Neurology, Daejeon, Republic of Korea

We report a 48-year-old lady who presented with one month history of dyspnea on exertion and orthopnea. Her cardiovascular and pulmonary examinations were unremarkable. On retrospect, she had a 4 year history of proximal lower limbs weakness with difficulty climbing upstair and gripping. The weakness appeared to be worsened following viral infection. She had no prior history of swallowing and breathing difficulty. However, she had noted frequent fall while running since age 22 but she had never seeking any medical advice. Her family history was unremarkable. On examination, she had high arch palate with mild facial weakness, symmetrical MRC grade 4/5 weakness on deltoid, biceps, triceps, iliopsoas, quadriceps, hamstring and tibialis anterior. Other muscles as well as deep tendon reflexes were normal. Her CK was 16 U/L. A needle EMG showed non-irritative myopathic pattern in the proximal and distal muscles of the arms and legs. Left vastus lateralis muscle biopsy shows some atrophic muscle fibre on H&E staining. Nemaline rod bodies in the subsarcolemmal area were found on modified Gomeri Trichrome stain and on electron microscopic study. doi:10.1016/j.nmd.2007.06.252

C.P.1.08 A homozygous deletion of TPM3 causing severe nemaline myopathy in two Turkish sib pairs from separate families Lehtokari, V. 1,*; Pelin, K. 2; Donner, K. 2; Voit, T. 3; RudnikScho¨neborn, S. 4; Wallgren-Pettersson, C. 1 1 Folkha¨lsan Institute of Genetics, University of Helsinki, Helsinki, Finland; 2 University of Helsinki, Helsinki, Finland; 3 Institut de Myologie, Paris, France; 4 RWTH Aachen University, Aachen, Germany The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders defined by structural abnormalities in the muscle fibres. Nemaline (rod) myopathy (NM) is a rare congenital myopathy diagnosed on the basis of muscle weakness and nemaline bodies in the muscle fibres. The nemaline bodies are protein aggregates derived from sarcomeric Z discs and thin filaments. The six known NM genes all encode proteins for the thin filament of the muscle sarcomere: nebulin, a-actin1, b- and c-tropomyosin, troponin T1 and cofilin 2. In order to identify the seventh NM gene we performed a genome-wide linkage study using microsatellite markers in 12 Turkish families with recessive NM. The search for the new gene is still ongoing, but in the context of this linkage study we found homozygosity at the TPM3 locus in two of the families. Sequencing of the TPM3 gene revealed a homozygous mutation in the affected sib pairs. The four children had severe forms of nemaline myopathy, with an unusual pectus carinatum

Background: Nemaline myopathy (NM) is a rare and clinically and genetically heterogeneous congenital myopathy, characterized by the presence of rod-like structures called nemaline (thread-like) bodies in the muscle fibers. It was world-wide distributed with incidence of 0.02 per 1000 live births. The clinical spectrum ranges from sever cases with antenatal or neonatal onset with early death to adult-onset cases with slow progression. To date mutations have been identified in five different genes, including ACTA1, NEB, TPM2, TPM3, and TNNT1. Objective: To investigate the clinicopathologic features of Korean patients with nemaline myopathy. Methods: This study is based on clinical, EMG, histochemical evaluation of seven patients at Severance Hospital, Yonsei University College of Medicine. The diagnosis was established by muscle biopsy. Results: Five male and two female patients were investigated. The onset of age is between infancy and 31 years (mean age: 9.7 years). Four patients had childhood onset, 1 patient had birth onset, and 1 patient had adult onset. The weakness was predominantly proximal in 6, equal proximally and distally in 1, and predominantly distal in 1. The initial weakness on onset was predominantly in lower extremities. The EMG showed myopathic MUAP in 5 and normal in 2. Of 5 patients with myopathic feature, two had denervating potentials. The serum CK level was normal in all patients. On light microscopy, the nemaline body was observed in type 1 and 2 fibers. All patients showed type 1 predominance and hypotrophy. Clinical course was slowly progressive or non-progressive in 6. But one patient had acute respiratory insufficiency. Conclusions: The seven Korean patients with nemaline myopathy had relatively heterogeneous clinical presentation from birth to adult onset, which is similar compared to other countries. doi:10.1016/j.nmd.2007.06.254

C.P.1.10 Molecular mechanisms underlying X-linked myotubular myopathy Sanoudou, D. 1; Kretz, C. 2; Beggs, A. 1; Laporte, J. 2; Buj-Bello, A. 2; Mandel, J. 2; Al-Qusairi, L. 2,* 1 Program in Genomics and Division of Genetics, Children’s Hospital Boston and Harvard Medical Sch, Boston, United States; 2 IGBMC, Department of Molecular Pathology, Strasbourg, France X-linked myotubular myopathy (XLMTM) is a severe congenital disease that affects the skeletal musculature leading to early postnatal death of most patients. The gene responsible for the disorder, MTM1, encodes a lipid phosphatase named myotubularin which dephosphorylates phosphoinositides (PtdIns3P and PtdIns3,5P2). Mtm1 knockout mice develop a progressive generalized myopathy with reduced life expectancy and

Abstracts / Neuromuscular Disorders 17 (2007) 764–900 present a muscle pathology that resembles that of XLMTM patients. The aim of our study is to identify the molecular cascades leading to XLMTM pathogenesis. For this, we have analyzed the transcriptome of Mtm1 knockout skeletal muscle before and after the appearance of pathological signs, at 2 and 6 weeks, respectively, by using Affymetrix whole genome arrays. Whereas no differences in gene expression were observed at 2 weeks of age, a highly active transcriptional response to myotubularin deficiency in muscles of 6-week-old mutants was detected, involving genes related to cell-surface receptor and intracellular signalling, vesicle trafficking, ion homeostasis, transcription, protein metabolism and muscle development. We focused our attention to genes involved in Ca2+ homeostasis, as this pathway has not been previously linked to the disease. We found that genes linked to the excitation–contraction coupling machinery are dysregulated at both the transcriptional (Q-PCR) and the protein level in muscle of 6-week-old mice. Interestingly, we found that the expression of several of these genes is already altered at early stages of the disease. In particular, we have identified two genes, Sln and Rrad, as early markers of murine XLMTM. These results suggest that defects in calcium handling may contribute to the pathogenesis of myotubular myopathy. Results on the investigation of calcium homeostasis will be presented. doi:10.1016/j.nmd.2007.06.255

C.P.1.11 Regulation of nebulin pre-mRNA splicing Ranta, S. *; Pelin, K. University of Helsinki, Department of Biological and Environmental Sciences, Helsinki, Finland Nebulin and its numerous isoforms are encoded by one gene, NEB, with 183 exons spanning 249 kb of genomic DNA on chromosome 2q22. Mutations in NEB cause autosomal recessive nemaline myopathy. Two regions in the central part and two in the 3 0 end of NEB harbours alternatively spliced exons. Based on the organization of the nebulin protein and our observations of the splicing pattern of NEB, we predict the theoretical number of different nebulin isoforms to be 3388. Objective: The aim of the project is to identify sequence elements and splice factors involved in the regulation of nebulin pre-mRNA splicing. Different computer programs were used to calculate splice site strengths and to predict splicing enhancer and silencer elements in NEB. The predicted elements are verified experimentally using co-transfection of NEB minigene expression vectors and antisense U7 small nuclear RNAs produced from the U7 smOpt plasmid in C2C12 cell cultures. The splicing is assessed by RT-PCR, agarose gel electrophoresis and sequencing. The role of different splice factors in the regulation of constitutive and alternative splicing of nebulin pre-mRNA is elucidated using siRNA knockdown of the splice factors in C2C12 cell cultures and the effect on Neb pre-mRNA splicing is assessed as described above. There is no significant difference in 5 0 - and 3 0 splice site strengths between the constitutively and alternatively spliced exons in the central region of NEB, but the alternative exons in the 3 0 end of NEB have significantly weaker 5 0 splice sites compared with all the other NEB exons. Only a subset of the predicted splicing regulatory elements in NEB serve as binding sites for splice factors in vitro. Identification of splicing regulatory elements is important for the interpretation of mutations and other sequence variants found in nemaline myopathy patients. doi:10.1016/j.nmd.2007.06.256

C.P.1.12 One causative gene, TPM2, and two congenital myopathies. A functional study Brudzewsky, D. 1,*; Marttila, M. 1; Nuutinen, E. 1; Ollila, S. 2; Donner, K. 3; Pelin, K. 2; Wallgren-Pettersson, C. 1

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University of Helsinki, Folkha¨lsan Institute of Genetics, Helsinki, Finland; 2 University of Helsinki, Department of Biological and Environmental Science, Helsinki, Finland; 3 University of Helsinki, Department of Medicine, Helsinki, Finland Tropomyosins together with the troponin complex regulate the binding of actin to myosin during muscle contraction. Mutations in the betatropomyosin (TPM2) gene have been reported in patients with distal arthrogryposis (R91G and R133W), nemaline myopathy (Q147P and E117K) and cap myopathy (Lehtokari et al., in press). There is an overlap between the three entities both clinically and histologically. The latter two entities are both classified as congenital myopathies and they share several clinical and histological features. They are distinguished on the basis of the formation of cap-like structures, with or without nemaline body formation. The aim of the study is to shed light on the pathogenetic pathways leading from the TPM2 mutations identified to the structural abnormalities seen in the muscle fibres and to clinical muscle weakness. Here, we study the aberrant protein products and their effects on the binding of beta-tropomyosin to actin, and on the ability of the tropomyosin molecules to form dimers. We initially used co-sedimentation of beta-tropomyosin proteins expressed in Escherichia coli to study tropomyosin-actin binding. To confirm the results obtained, suggesting altered affinity, we are now expressing normal and aberrant variants of beta-tropomyosin proteins in a baculovirus expression system. This ensures acetylation of the NH2 terminus, which is important for the affinity of beta-tropomyosin for actin, and for the dimerisation of tropomyosin molecules. Actin binding is monitored by an in vitro sedimentation assay, and the formation of homo- or hetero-dimers is evaluated using Western blot analysis. Our preliminary results suggest that the alterations of the beta-tropomyosin proteins caused by the TPM2 mutations identified affect the binding of tropomyosin to actin. The study is ongoing. doi:10.1016/j.nmd.2007.06.257

C.P.1.13 A homozygous null mutation in TPM2 gene causes autosomal recessive nemaline myopathy associated with multiple pterygia Jouk, P.; Labarre-Vila, A.; Mezin, P.; Drouhin, S.; Marty, I.; Lunardi, J.; Monnier, N. * Centre de re´fe´rence des maladies neuromusculaires, CHU Grenoble, Grenoble, France Tropomyosins (TM) are a family of highly conserved actin-binding proteins composed of two subunits that form a coiled-coil structure. Skeletal muscle contains two forms of tropomyosin (alpha and beta subunits) localized to the thin filaments where they regulate Ca2+-dependant muscle contraction in association with the troponin complex. The beta subunit is encoded by the TPM2 gene composed of 9 exons of which four are alternatively spliced (exons 1, 2, 6 and 9). Mutations in TPM2 gene have been associated so far with two allelic neuromuscular disorders, autosomal dominant nemaline myopathy (NEM) and distal arthrogryposis (DA). We report an Algerian consanguineous family in which a male child presented at birth with severe hypotonia, distal amyotrophy, arthrogryposis and multiple pterygia, facial dysmorphia, ptosis and ophtalmoplegia. Three first cousins were also affected. Nemaline myopathy was diagnosed on muscle biopsy. After exclusion of ACTA1, TPM3 and NEB genes by linkage studies, a null mutation was identified at a homozygous level in all affected family members. The mutation is localized in exon 6b, specific of the skeletal muscle isoform. A low amount of transcript was detected by quantitative RT-PCR and absence of TPM2 expression in the muscle was confirmed by Western blot analysis. Identification of a null allele mutation in the TNNT1 gene has been reported in the Amish population in association with a lethal form of autosomal recessive nemaline myopathy. By contrast, the null mutation identified in the family is not life-threatening